Background:Spondiaspinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract. Objective: Since its bioactive compounds are largely unknown, this study was designed to characterize the lead components present in it and their anticancer activity against human glioblastoma cell line (U87). MaterialsandMethods: Major compounds from the ethyl acetate fraction were isolated by column chromatography and their anticancer potentials against U87 cells were evaluated. Furthermore, flow cytometric and immunoblotting analyses were performed to demonstrate the mechanism of apoptosis inducing activity of methyl gallate (MG) against U87 cell line. Results: Four major compounds were isolated from the ethyl acetate fraction. Amongst these, two compounds showed promising activities and with the help of different spectroscopic methods they were identified as gallic acid and MG. Flow cytometric studies revealed that MG-induced apoptosis in U87 cells dose-dependently; the same was confirmed by activation of caspases through cleavage of endogenous substrate poly (adenosine diphosphate-ribose) polymerase. MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression. Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38). Conclusion: MG is a potent antioxidant and it induces sustained ERK1/2 activation and apoptosis in human glioblastoma U87, and provide a rationale for evaluation of MG for other brain carcinoma cell lines for the advancement of glioblastoma therapy.

Cancer is a universal public health crisis because of its high occurrence and mortality rate. Regardless of recent advances in the development of new diagnostic and therapeutic tools, it remains of some urgency to identify new and better therapeutic agents and regimes for cancer. Bioactive natural products provide structures that will become major sources of novel agents with pharmacological promise. [1] Glioma is the most common and highly malignant type of primary brain tumor in humans [2] which compared to other cancers, is relatively rare but their high malignancy rate make them responsible for a very high mortality rate. Despite invasive surgery, radiotherapy and chemotherapy, malignant brain tumors remain a therapeutic challenge, [3] due to its characteristic resistance to apoptosis. [4] Doxorubicin, a well-known chemotherapeutic drug used in treating gliomas has some serious side effects that are responsible for congestive heart failure. [5] Thus, major attention is being given to search for better and safer antioxidants of natural origin, which may raise the efficiency of cancer treatment.

Spondiaspinnata (Linn. f.) Kurz. (Family - Anacardiaceae) is a deciduous tree widely distributed in South-East Asian countries. The plant is cultivated in various parts of India as the fruits are eaten as a vegetable when green and as a fruit when ripe and local people make chutney, jam and pickle. Fruits are very nutritious and rich in vitamin A, minerals and iron content. [6] Ethnomedically, S.pinnata is used for its antibacterial activity [7] and treatment of dysentery. [8] It is also reported that the bark of S.pinnata acts as a very good antioxidant and free radical scavenger [9] as well as a potent iron chelator. [10] Our recent findings suggest that the bark of S.pinnata also possesses promising anticancer activity. [11] The gum exudate of the species has been reported to contain acidic polysaccharides. [12] Oleanolic acid has been isolated from the fruit of this species, and its antimicrobial properties have been established previously. [13] These findings further culminate our interest in isolating and characterizing the active principles from S.pinnata and investigated for their various pharmacological potentials like anticancer activity.

Cell viability was quantified using the WST-1 cell proliferation reagent, Roche diagnostics, according to the previously described method. [11] In brief, 1 × 10 4 cells were treated with compounds ranging from 1 to 30 μg/ml for 48 h in 96-well culture plate. After treatment, 10 μl of WST-1 cell proliferation reagent was added to each well followed by 2 h of incubation at 37°C. Cell viability was quantified by measuring absorbance at 460 nm using a microplate enzyme-linked immunosorbent assay reader MULTISKAN EX (Thermo Electron Corporation, USA).

Cell cycle analysis was performed by flow cytometry using the method previously described. [11] U87 cells (1 × 10 5 ) were treated with methyl gallate (MG) ranging from 10 to 100 μM for 24 h. After treatment, nuclear DNA of cells was then stained with propidium iodide (PI) and cell phase distribution was determined on FACSVerse (Becton Dickinson) equipped with 488 nm (blue), 405 nm (violet) and 640 nm (red) solid state laser light using FACSuite software version 1.0.3.2942 (Becton Dickinson). A total 10,000 events were acquired, and data analysis was done using the same software. A histogram of DNA content (x-axis, red fluorescence) versus count (y-axis) was plotted.

Annexin V staining

This assay was performed using Annexin-V-FLUOS Staining kit, Roche diagnostics. U87 cells (1 × 10 5 ) were treated with MG ranging from 10 to 100 μM for 24 h. After treatment cells were labeled with PI and FITC according to the protocol of the kit manufacturer. The distribution of apoptotic cells was identified by flow cytometer on FACSVerse (Becton Dickinson) equipped with 488 nm (blue), 405 nm (violet) and 640 nm (red) solid state laser light using FACSuite software version 1.0.3.2942 (Becton Dickinson). A total 10,000 events were counted. Cells that were Annexin V (−) and PI (−) were considered as viable cells. Cells that were Annexin V (+) and PI (−) were considered as early stage apoptotic cells. Cells that were Annexin V (+) and PI (+) were considered as late apoptotic or necrotic cells.

Western blot analysis

1 × 10 6 U87 cells were treated with MG (50 μM) for various time intervals (3-36 h). After treatment, cells were lysed with triple detergent cell lysis buffer (50 mM tris-Cl, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate (SDS), 1% triton X-100, 0.5% sodium deoxycholate, 1 μg/ml aprotinin, 100 μg/ml phenyl methyl-sulfonyl fluoride, pH 8) and the lysates were then centrifuged at 13,800 ×g for 20 min at 4°C. Protein concentration was measured by Folin-Lowry method. Proteins (40 μg) in the cell lysates were resolved on 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane and the membranes were then blocked with 5% nonfat dry milk in tris-buffered saline (TBS). After overnight blocking, the membranes are incubated for 3 h with corresponding antibodies. Postwash with TBS-T (0.01% of Tween-20 in TBS) membranes were incubated with alkaline phosphatase conjugated anti-Rabbit immunoglobulin G antibody at room temperature in the dark for 4 h, followed by washing. The blots were then developed with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate, and the images were taken by the imaging system EC3 Chemi HR (UVP, USA). The blots were then analyzed for band densities using ImageJ 1.45s software (National Institute of Health, USA).

For the past 30 years, the emphasis in natural product research has been in the field of anticancer drugs. [13],[14] The traditional medicinal use of S.pinnata was previously supported with the finding of antioxidant, hepatoprotective and anticancer properties of this plant. So, the current study focuses on the bioactivity guided isolation and characterization of the active principles from S.pinnata bark and since our preceding studies showed effective antioxidant and anticancer activities with the ethyl acetate fraction of 70% methanolic extract of S.pinnata bark, this fraction was subjected to different isolation methods and four major compounds were isolated (SPE1, SPE2, SPE3 and SPE4) and used for further anticancer investigations.

The cytotoxic activity of the four isolated compounds against U87 cells is shown in [Figure 1]. Results suggest that SPE3 and SPE4 showed significant toxicity against U87 cells dose-dependently with an IC 50 value 59.28 ± 3.92 μg/ml and 8.44 ± 0.61 μg/ml respectively while SPE1, SPE2 showed negligible toxicity in any of the examined concentrations with a very high IC 50 value of 841.54 ± 89.32 μg/ml and 280.42 ± 34.89 μg/ml respectively. As a whole, we can say that SPE3 and SPE4 are the lead compounds as an anticancer agent in ethyl acetate fraction of S.pinnata bark. These two purified compounds are then subjected for their structural elucidation using various spectroscopic methods which indicated SPE3 to be gallic acid [Figure 2]a and SPE4 as MG [Figure 2]b. The spectroscopic data are shown as follows:

Gallic acid is a phenolic compound and previously reported effective against many types of the malignancies [15] including various gliomas especially U87. [16] MG is a derivative of gallic acid. The anticancer properties of MG are not widely proven rather there are some reports regarding its antitumor [17] and proliferation inhibition activities. [18] However, its biological activity has not been extensively examined in glioma and other cancer cells. With the interest of revealing the underlying mechanism of MG-induced cytotoxicity against U87, further flow cytometric as well as immunoblotting studies were carried out. The effect of MG on cell cycle distribution of U87 cells resulted in an increase in accumulation of Sub-G1 phase that refers to the apoptotic cells [Figure 3]. The apoptosis was found to increase dose-dependently with nearly 40% cells in sub-G1 at a concentration of 100 μM. Furthermore, the dose-dependent induction of apoptosis in MG treated U87 cells was confirmed by Annexin-V staining [Figure 4]. The results showed an increase in early apoptotic cells with an increase in dose of MG. At zero dose of MG, 4.12% cells were found in early apoptotic phase while 4.46%, 5.63%, 28.12%, 43.63% and 50.08% cells were found in early apoptotic phase with an increase in doses 10 μM, 25 μM, 50 μM, 75 μM and 100 μM respectively suggesting that the MG treatment induced apoptosis but not necrosis in U87 cells.

Mitochondria play a pivotal role in the activation of caspase cascade and thereby signal transduction of apoptosis. [19] Effects of MG on the proteolytic activation of caspase-9 and caspase-3 were then examined. As shown in [Figure 5]a and b, MG treatment resulted in a significant increase in the active form of caspase-9 and caspase-3 in U87 cells. Furthermore, procaspse-8 level decreased, caspase-8 level elevated [Figure 5]c. The activation of caspases in MG treated U87 cells was further confirmed by detecting the cleavage of PARP, an endogenous substrate of activated caspase-3 and a hallmark of apoptosis. As shown in [Figure 5]d, treatment of U87 cells with MG resulted in the cleavage of PARP to a 25 kDa fragment. The observation of MG-mediated activation of caspase-9, caspase-3, subsequent cleavage of PARP in U87 cells, suggesting that mitochondrial-mediated caspase cascade pathway plays a very important role in MG-induced apoptosis in U87 cells. [20] Bcl-2 family proteins, including Bcl-2 and Bcl-2-related family members such as Bax, Bid, Bcl-extra-large and Bcl-2-associated death promoter, play an important role in the regulation of apoptosis. The balance between the expression levels of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), is critical for cell survival and death as the increase in Bax/Bcl-2 ratio contributes to the release of cytochrome C from mitochondria and decides the susceptibility of cells to undergo apoptosis. [21] As shown in [Figure 6]a, MG treatment increased the level of Bax expression while reduced Bcl-2 expression in a time-dependent manner. A densitometric analysis of the bands revealed that MG increased the Bax/Bcl-2 ratio, which is responsible for MG-induced apoptosis in U87 cells. As cleaved caspase-8 level was elevated there was an appearance of the t-Bid [Figure 6]b after the treatment with MG. Previously it is reported that, caspase-8 links intrinsic pathway with extrinsic pathway by cleaving Bid into truncated-Bid and plays an important role in activation of both these pathways. [22] It is possible that p53 molecule plays a role in the alteration of the expression of Bax and Bcl-2. Our results showed that MG treatment induces the expression of p53 in U87 cells [Figure 7]a. This suggests that p53 is responsible for the upregulation of Bax and down regulation of Bcl-2 in MG treated U87 cells. The ability of wild type p53 to upregulate Bax and downregulate Bcl-2 and proceed to the apoptosis is demonstrated previously. [23]

Mitogen-activated protein kinases (MAPK) (extracellular signal-regulated kinase 1/2 [ERK 1/2], c-Jun N-terminal of stress-activated protein kinases 1/2 [JNK1/2] and the p38 protein kinases) participate in diverse cellular functions such as cell proliferation, cell differentiation, cell motility and cell death. Cellular stresses and stimuli induce cell apoptosis via sustained activation of the MAPK signaling pathways. [24] We thus examined the effect of MG treatment on activation of ERK1/2, JNK and p38 in U87 cells. The phosphorylation level of ERK1/2 was increased [Figure 7]b] after MG treatment, which lasted 18 h. However, no significant changes in phosphorylation levels of JNK and p38 were observed [Figure 7]c and d. It was proved that the transient activation of ERK1/2 plays an essential role in cell proliferation, and that sustained ERK1/2 activation induces apoptosis. [25] These results suggested that sustained activation of the ERK is involved in MG-induced growth inhibition and apoptosis in U87 cells.

Conclusion

We can say that MG, isolated for the first time from S.pinnata bark, inhibited cell growth and induced cell apoptosis in glioma cells. The further western blotting studies revealed the underlying principle of MG-induced apoptosis in glioma cells that involve the activation of caspase-3 and caspase-9 and Bid truncation. We found that increased p53 level, Bax/Bcl-2 ratio and sustained ERK1/2 activation were also associated factors for MG-induced apoptosis [Figure 8]. The results provide a rationale that MG can be developed as a potential anticancer agent against human glioblastoma. Furthermore, extensive in vitro studies against different glioblastoma cell line and in vivo research with xenograft and preclinical models with MG will justify its use as potent drug, thus making it a promising candidate for management of brain cancer patients.

Acknowledgments

The authors would like to acknowledge Council of Scientific and Industrial Research, Govt. of India for providing the necessary funds to conduct the study. Acknowledgments are also due to Mr. Ranjit K. Das, Mr. Pradip K. Mallick, Mr. Ranjan Dutta (CIF, Bose Institute) and Mr. Barun Majumder (CIF, Bose Institute) for their technical assistance.